Integral membrane proteins represent about 30% of the coding sequences of eukaryotic genomes. Their functions, and especially those of these proteins which are inserted into the cell plasma membrane and exposed to the outside environment, have such an importance that they are privileged targets in the fields of biomedical sciences and pharmacology. They are in fact thought to be the target for at least 60% of the therapeutic agents currently commercially available. Membrane proteins serve in particular as binding sites, attachment points or privileged targets for multiple interactions. These range from recognizing small ligands, such as neurotransmitters or hormones, to cell association into tissues. Membrane proteins are further most often the first target for viruses, pathogenic bacteria or parasites, or antibodies during immune defense or autoimmune diseases. They can also be involved in membrane/DNA or membrane/cytoskeleton association, in regulation or deregulation of cell division (cancers), and they are recognized by macromolecular effectors such as G proteins or kinesins.
Due to their importance in the fields of biomedical sciences and pharmacology, it is absolutely essential to provide tools for studying membrane proteins and their ligands. In particular, it is important to be able to detect ligand binding to a membrane protein of interest. In fact, a process for detecting ligand binding has several very valuable applications:                in the case where the membrane protein is derived from the membrane of a pathogenic agent, such a process may be useful to detect in a biological sample obtained from a subject the presence or absence of antibodies raised against this membrane protein, and accordingly the presence or absence of an exposition of the subject to the pathogenic agent;        in the case of a human or animal membrane receptor shown to be involved in the pathogenesis of a disease and therefore providing a therapeutic target for the treatment of such disease, such a process may be useful to screen compound libraries so as to identify agonist or antagonist compounds for this membrane receptor.        
To carry out a process for detecting ligand binding to a membrane protein, it is very useful to have this membrane protein immobilized onto a support. It has been long known how to attach or immobilize soluble proteins onto supports. However, the problem is more complex for membrane proteins. In fact, membrane proteins necessarily expose highly hydrophobic surfaces that, in situ, interact with the membrane, which makes their handling difficult, particularly in that it is generally necessary to use high amounts of detergents to isolate them from the membrane and to make them soluble. Compared to cytosolic proteins, their handling and accordingly their immobilization on a support is made a lot more strenuous due to their hydrophobicity.
Different immobilization techniques have been developed to deposit membrane proteins on the surface of a support. For example, by genetically or chemically modifying the protein, a functional group can be inserted at one of the extremities of the protein polypeptide chain so as to promote protein adhesion to the surface of a functionalized support (for example, by introducing a terminal His-Tag on the protein, which will interact with a support grafted with NTA groups coordinating Ni2+ or Co2+ ions) (1.2).
However, such a technique relies on the possibility of genetically or chemically modifying the protein of interest, and is thus difficult or impossible to implement for a membrane protein about which little information is available. Furthermore, since genetically or chemically modifying a protein involves a number of steps this can be cumbersome and tedious. Lastly, its development should be repeated for each protein of interest and accordingly this technique cannot be used either routinely or for a large number of membrane proteins simultaneously.
In another technique, it is possible, by maintaining the protein in its original cell membrane, or by inserting it into an artificial membrane (for example, a vesicle), to promote protein adhesion onto a support grafted with hydrophobic chains via interactions between the chains and the membrane (3-5). A further technique comprises utilizing the charge properties of extra-membrane domains of the protein, or hydrophilic heads of lipids of the membrane in which the protein is inserted, to promote simple electrostatic interactions with a charged support.
However, for both these techniques, either the protein is extracted from the membrane, and it is generally absolutely necessary to work in the presence of a detergent, which makes the immobilization methods considerably more complex, both because the solution properties are altered (for example by reducing the surface tension of the solutions, which can make the spotting of the proteins at the surface of the chips inaccurate) and because the presence of detergent affects the stability of a number of membrane proteins (particularly membrane complexes), or proteins inserted into natural or synthetic membranes (lipid vesicles) are used, and the technique is thus intricate and raises a sensitivity problem: the low density of the protein of interest may decrease the signal/noise ratio of the experiment, and the presence of undesirable components in the case of natural membranes (other proteins, a large variety of natural lipids, various cofactors) may introduce an interference between the experimental signal and adverse side reactions and background noise.
As a result, it is clear that the existing immobilization (or attachment) methods of membrane proteins onto supports are unsatisfactory. There is therefore a need for a process for attaching membrane proteins on supports which overcomes the above-mentioned drawbacks, i.e. for a process having the following characteristics:                a process for maintaining membrane proteins in a completely detergent-free water-soluble and biochemically stabilized form, thus making the method simpler and preventing membrane proteins from destabilizing,        a universal process, applicable to any membrane protein, without any particular adjustment of the experimental method to each protein and without the need for any protein modification, which can thus be used including when the protein is not fully known or when its biochemistry and its genetics are not controlled, and        a process for attaching membrane proteins to the surface of a support with a high density, resulting in better yields and a better sensitivity of the experimental results and accordingly a better interpretation of the data.        
Amphipols are polymeric amphiphiles having a good solubility, a number of hydrophobic side chains, and molecular dimensions and flexibility so as to enable them to combine at multiple points with the hydrophobic transmembrane surface of membrane proteins (FIG. 1, references 6, 7). The principle of multi-point attachment is to guarantee that desorption kinetics is very slow, so as to make the combination between the membrane protein and the amphipol essentially irreversible.
Furthermore, it has been shown that this approach is universal, which means that all membrane proteins tested to date, i.e. more than twenty, can be maintained in solution in the form of amphipol complexes (7, 8). Furthermore, the resulting trapped proteins maintain their native structure, remain soluble in the absence of free surfactant in the solution (7-9), and their stability is at worst matched but most often improved compared to keeping in detergent solution (7, 8, 10). Lastly, when a mixture of membrane proteins is trapped by amphipols under appropriate conditions, all membrane proteins in the mixture are trapped separately (8).
Amphipols are therefore a tool for stabilizing in a solution any membrane protein, irrespective of its structure, function and/or origin. However, they have only been used to date for solubilizing membrane proteins and for engineering them in a solution, or for stabilizing them temporarily, and no study has ever been disclosed as to the possibility of further using them as intermediates mediating membrane protein attachment to a support. On the contrary, up to now, amphipols have only been used in order to provide water-soluble complexes, that can move freely in a solution, from membrane proteins naturally insoluble in aqueous media, for temporarily stabilizing such membrane proteins in lipid membrane regeneration experiments (11), or for stabilizing such biotinylated membrane proteins attached through usual biotin/avidin interaction on a solid support, with amphipol only being used to stabilize the protein and not being at all involved in the combination of the membrane protein to the solid support (8).